Fish Physiolcgy and Biochemistry vol. 12 no. 3 pp 221-235 (1993) Kugler Publications, Amsterdam/New York

Essential fatty acid requirement of juvenile red drum (Sciaenops ocellatus) Rebecca T. Lochmann and Delbert M. Gatlin III Department of Wildlife and FisheriesSciences, Texas A&M University System, College Station, TX 77843-2258 U.S.A. Accepted: May 7, 1993 Keywords: red drum, Sciaenops ocellatus, essential fatty acids, (n-3) highly unsaturated fatty acids, lipid nutrition, fatty acid composition

Abstract Feeding experiments and laboratory analyses were conducted to establish the essential fatty acid (EFA) requirement of red drum (Sciaenops ocellatus). Juvenile red drum were maintained in aquaria containing brackish water (5 ± 2%0 total dissolved solids) for two 6-week experiments. Semipurified diets contained a total of 7% lipid consisting of different combinations of tristearin [predominantly 18:0] and the following fatty acid ethyl esters: oleate, linoleate, linolenate, and a mixture of highly unsaturated fatty acids (HUFA) containing approximately 60% eicosapentaenoate plus docosahexaenoate. EFA-deficient diets (containing only tristearin or oleate) rapidly reduced fish growth and feed efficiency, and increased mortality. Fin erosion and a "shock syndrome" also occurred in association with EFA deficiency. Of the diets containing fatty acid ethyl esters, those with 0.5-1% (n-3) HUFA (0.3-0.6% eicosapentaenoate plus docosahexaenoate) promoted the best growth, survival, and feed efficiency; however, the control diet containing 7% menhaden fish oil provided the best performance. Excess (n-3) HUFA suppressed fish weight gain; suppression became evident at 1.5% (n-3) HUFA, and was pronounced at 2.5%. Fatty acid compositions of whole-body, muscle and liver tissues from red drum fed the various diets generally reflected dietary fatty acids, but modifications of these patterns also were evident. Levels of saturated fatty acids appeared to be regulated independent of diet. In fish fed EFA-deficient diets (containing only tristearin or oleate), monoenes increased and (n-3) HUFA were preferentially conserved in polar lipid fractions. Eicosatrienoic acid [20:3(n-9)] was not elevated in EFA-deficient red drum, apparently due to their limited ability to transform fatty acids. Red drum exhibited some limited ability to elongate and desaturate linoleic acid [18:2(n-6)] and linolenic acid [18:3(n-3)]; however, metabolism of 18:3(n-3) did not generally result in increased tissue levels of (n-3) HUFA. Based on these responses, the red drum required approximately 0.5% (n-3) HUFA in the diet (approximately 7% of dietary lipid) for proper growth and health. Introduction The aquacultural potential of the red drum (Sciaenops ocellatus), a euryhaline sciaenid, is currently constrained by several factors including a lack of sufficient information on this species' nutri-

tional requirements, especially dietary lipid and essential fatty acid (EFA) requirements. Qualitative and quantitative EFA requirements vary markedly in different fish species (Watanabe 1982). When appropriate essential fatty acids are not available in sufficient quantities poor growth and feed efficien-

Correspondenceto: Delbert Gatlin, Dept. of Wildlife and Fisheries Sciences, Texas A&M University, College Station, TX 77843-2258; phone: 409/847-9333; FAX: 409/845-4096

222 Table 1. Ingredient (% dry weight) and analyzed fatty acid (% of total lipid) composition of diets in experiment 1 Diet number Ingredient/fatty acid

1

2

3

4

5

6

7

8

9

Tristearinl Oleatel Linoleate' Linolenatel (n-3) HUFA mix2 Menhaden fish oil 3 Amino acid premix 4 ,5 Extracted drum muscle 6 Dextrin 5 Cellulose 5 Carboxymethyl cellulose 5 Vitamin premix 7 Mineral premix 7 Ethoxyquin'

7.0 0.0 0.0 0.0 0.0 0.0 17.0 19.1 37.3 9.8 2.0 3.0 4.0 0.013

6.0 1.0 0.0 0.0 0.0 0.0 17.0 19.1 37.3 9.8 2.0 3.0 4.0 0.013

5.0 2.0 0.0 0.0 0.0 0.0 17.0 19.1 37.3 9.8 2.0 3.0 4.0 0.013

6.0 0.0 1.0 0.0 0.0 0.0 17.0 19.1 37.3 9.8 2.0 3.0 4.0 0.013

5.0 0.0 2.0 0.0 0.0 0.0 17.0 19.1 37.3 9.8 2.0 3.0 4.0 0.013

6.0 0.0 0.0 1.0 0.0 0.0 17.0 19.1 37.3 9.8 2.0 3.0 4.0 0.013

5.0 0.0 0.0 2.0 0.0 0.0 17.0 19.1 37.3 9.8 2.0 3.0 4.0 0.013

5.0 0.0 1.0 1.0 0.0 0.0 17.0 19.1 37.3 9.8 2.0 3.0 4.0 0.013

6.0 0.0 0.0 0.0 1.0 0.0 17.0 19.1 37.3 9.8 2.0 3.0 4.0 0.013

0.0 0.0 0.0 0.0 0.0 7.0 17.0 19.1 37.3 9.8 2.0 3.0 4.0 0.013

16:0 18:0

3.4 79.9

5.9 21.1

3.9 9.0

5.5 22.2

3.6 16.9

4.6 36.3

3.4 23.6

3.8 22.4

5.2 62.9

14.2 5.4

18:1

0.5

48.3

64.4

-8

-

-

-

-

-

10.1

18:2(n-6) 18:3(n-3)

0.4 -

1.8 0.8

1.6 0.3

52.0 1.3

63.2 0.1

2.8 37.6

2.4 59.4

29.1 30.6

18:4(n-3)

-

0.9

0.3

-

0.9

-

-

-

2.0

3.5

20:5(n-3)

-

-

-

-

-

-

-

-

10.8

13.3

22:6(n-3)

-

-

-

-

-

-

-

-

8.0

15.6

2.0 0.3

10

2.4 1.8

l Purchased from Sigma Chemical Co., St. Louis, MO; 2 the (n-3) HUFA mix was a combination of highly unsaturated fatty acid ethyl esters that consisted predominantly of 20:5(n-3) and 22:6(n-3) (60% total). This mixture was obtained from the Charleston Laboratory of the National Oceanographic and Atmospheric Administration (NOAA); 3 winterized (cold-pressed) product donated by Zapata Haynie Corporation, Reedville, VA; 4 the amino acid premix, consisting of the L-isomers of crystalline amino acids, had the following composition (%): arginine HCI, 8.24; glycine, 5.54; histidine, 2.84; isoleucine, 5.66; leucine, 9.26; lysine HCI, 14.15; methionine, 3.73; cystine, 3.47; phenylalanine, 4.63; tyrosine, 4.12; serine, 4.44; threonine, 5.28; tryptophan, 0.97; valine, 6.18; proline, 10.75; alanine, 10.75; 5 purchased from United States Biochemical Corporation, Cleveland, OH; 6 fish muscle consisted of a mixture of black drum and red drum which was solvent-extracted to remove extraneous lipid as described in methods; 7 same as Moon and Gatlin (1991); 8 not detected.

cy generally occur. Other deficiency signs such as eroded caudal fin, "shock syndrome", and fatty livers have been observed in rainbow trout (Oncorhynchus mykiss) and other fish species (Watanabe 1982). Fatty acid profiles of fish tissue in comparison with dietary fatty acids generally provide additional information on how these nutrients are utilized, and facilitate further characterization of EFA requirements. The EFA requirement of red drum was expected to parallel those of other marine carnivorous fishes such as the red sea bream (Chrysophrys major) which have a limited ability to elongate and desaturate linolenic acid, and thus, require dietary (n-3) highly unsaturated fatty acids (HUFA) (Yone

1978; Watanabe 1982). To investigate this hypothesis, feeding experiments and laboratory analyses were conducted to establish the EFA requirement of red drum.

Materials and methods Experimental diets The composition of semipurified diets for experiments 1 and 2 are shown in Tables 1 and 2, respectively. All diets contained 357o crude protein and 3.6 kcal estimated available energy/g (Serrano et al. 1992). Dietary protein was provided by both crys-

223 Table 2. Ingredient (o

dry weight) and analyzed fatty acid (

of total lipid) composition of diets in experiment 2 Diet number

Ingredient/fatty acid

11

12

13

14

15

16

Tristearin1 Linolenatel (n-3) HUFA mix 2 Menhaden fish oil 3 Amino acid premix 4 ,5 Extracted drum muscle6 Dextrin 5 Cellulose 5 Carboxymethyl cellulose 5 Vitamin premix 7 Mineral premix 7 Ethoxyquinl

7.0 0.0 0.0 0.0 17.0 18.5 37.3 9.8 2.0 3.0 4.0 0.013

6.5 0.0 0.5 0.0 17.0 18.5 37.3 9.8 2.0 3.0 4.0 0.013

5.5 0.0 1.5 0.0 17.0 18.5 37.3 9.8 2.0 3.0 4.0 0.013

4.5 0.0 2.5 0.0 17.0 18.5 37.3 9.8 2.0 3.0 4.0 0.013

5.0 2.0 0.0 0.0 17.0 18.5 37.3 9.8 2.0 3.0 4.0 0.013

0.0 0.0 0.0 7.0 17.0 18.5 37.3 9.8 2.0 3.0 4.0 0.013

16:0 18:0 18:1 18:2(n-6) 18:3(n-3) 18:4(n-3)

3.4 79.9 0.5 0.4 -

2.7 82.9 0.2 0.3

2.5 55.8 0.3 0.4 0.2 3.7

1.5 27.9 0.6 0.6 0.3 6.7

3.4 23.6 _8 2.4 59.4 -

14.2 5.4 10.1 2.4 1.8 3.5

20:5(n-3)

-

5.9

14.9

28.8

-

13.3

22:6(n-3)

-

1.1

11.2

22.8

-

15.6

0.3

' All superscripts refer to the footnotes in Table 1.

talline amino acids and a mixture of red drum and black drum (Pogonius chromis) muscle. Previous research in this laboratory indicated that red drum achieved satisfactory growth when fed fish muscle protein in combination with crystalline amino acids. Prior to incorporation into diets, the drum muscle was freeze-dried and solvent-extracted with hexane and 95% ethanol to reduce lipid content to approximately 0.3% of dry weight. All diets were formulated to contain 7% total lipid which was provided by different combinations of specified fatty acid ethyl esters and triglycerides. The diets containing 7% tristearin (diets 1 and 11) in both experiments consisted of predominantly stearate (18:0) which does not have EFA activity in vertebrates since they can synthesize this fatty acid de novo. Therefore, tristearin served as a base lipid to which different fatty acid ethyl esters were supplemented to test their individual effects. The diets containing menhaden fish oil (diets 10 and 16) were used as controls since red drum have been shown to perform well with this dietary lipid (Williams and Robinson 1988). The remaining diets in experiment

I (diets 2-9) contained 1 or 2% levels of oleate (18:1), linoleate [18:2(n-6)], linolenate [18:3(n-3)], a mixture of 1% each of linoleate and linolenate, or 1% HUFA of the (n-3) series. The (n-3) HUFA consisted of ethyl esters that contained about 60% eicosapentaenoate [20:5(n-3)] plus docosahexaenoate [22:6(n-3)]. Based on the results of experiment 1, additional treatments in experiment 2 were chosen to define the requirements of juvenile red drum for (n-3) HUFA more precisely using 0.5, 1.5, or 2.5% levels (diets 12-14), and to re-examine the apparent partial EFA activity of linolenate at a 2% level (diet 15). The 7% tristearin and menhaden oil diets were used again as EFA-deficient and control diets, respectively. Procedures for diet preparation were as previously described (McClain and Gatlin 1988). In addition, all diets were supplemented with 125 mg ethoxyquin/kg. Diets were freeze-dried to reduce moisture without degrading lipids and stored at -18 0 C in air-tight bags until needed. Small quantities were refrigerated (4°C) in covered containers for no more than 3 d prior to feeding.

224 Culture system and experimental design Juvenile red drum originating from the Aquacultural Research and Teaching Facility of the Texas A&M University System were exposed to similar environmental conditions for the duration of both feeding experiments. The fish were maintained in a recirculating, brackish-water system at a salinity of 5 ± 2%0 total dissolved solids and a temperature of 25 2°C. The water was prepared by mixing well water with synthetic sea salts and was supplied to individual 110-1 aquaria at 700 ml/min. This lowsalinity water is beneficial to red drum (Gatlin et al. 1992) and conserves the use of artificial sea salts. Water quality was monitored and maintained by biofiltration, aeration, and mechanical filtration. Prior to the beginning of each experiment, all fish were fed the diet containing 7% tristearin for 2 weeks while adjusting to culture conditions. During this conditioning period the fish also were partially depleted of tissue fatty acid reserves because they were consuming a diet known to be deficient in essential fatty acids. This procedure was employed to minimize the influence of previous nutritional status of the fish on treatment effects. After the conditioning period, fish initially averaging 0.92 g each were placed in individual aquaria as groups of 20 fish with a total weight of 18 +±0.9 g (mean ± SEM) in experiment 1. In experiment 2, fish initially averaging 2.98 g were placed into individual aquaria as groups of 14 fish with a total weight of 42 ± 2.3 g. Each diet was fed to three replicate groups of fish for 6 weeks in both experiments. Both experiments were terminated at 6 weeks because high mortality had occurred in fish fed 7% tristearin and other treatment differences also were evident. During the experimental periods fish were fed 5-7% of their body weight daily divided into two equal feedings. All experimental fish groups were weighed weekly and mortalities were recorded. Mortalities were replaced during the first week of each experiment to compensate for possible stress effects due to handling during stocking procedures. Sample collection and analyses Fish were collected and frozen in water for subsequent analyses at the end of each feeding trial.

Pooled whole-body samples from each group consisting of 2-6 fish were finely minced with a cleaver prior to analyses. Samples consisting of 2-6 additional fish per group provided muscle and liver tissues. Thus, three composite samples per tissue per dietary treatment were analyzed and the results averaged for each tissue-lipid fraction. Samples were extracted using chloroform and methanol (2:1, v/v) with a Polytron homogenizer. An aliquot of the chloroform/methanol extract was taken to determine total lipid gravimetrically (Folch et al. 1957). Remaining lipid extract was reserved for fatty acid analysis. Polar and neutral lipids were separated using silicic acid columns. Columns were washed with 40 ml of hexane:ethyl ether (1:1, v/v) to collect neutral lipid, followed by 20 ml each of methanol and chloroform:methanol (1:1, v/v) to elute polar lipid. Polar and neutral lipids were transesterified with boron trifluoride. Fatty acid methyl esters were analyzed with a Varian 3400 gas chromatograph equipped with a 30-m x 1.53-mm SupelcowaxTM fused silica capillary column and a flame-ionization detector. Helium was used as the carrier gas. Fatty acids were identified by comparison of retention times to those of known standards and expressed as a percent of total fatty acids. Statisticalanalysis Treatment means for weight gain, survival, feed efficiency, and fatty acid concentrations of wholebody, muscle and liver were compared using the General Linear Models procedure of SAS (SAS Institute 1985) for a one-way analysis of variance. Duncan's multiple range test was used to test for differences among treatment means at alpha= 0.05. Results Experiment I Performance The lowest weight gain occurred in fish fed EFAdeficient diets containing 7% tristearin or oleate (Table 3). This growth depression was evident as early as after the first week of feeding. The addition of linoleate, linolenate or both increased fish

225 Table 3. Performance of juvenile red drum fed diets differing in fatty acid content for 6 weeks (experiment

Diet number 1 2 3 4 5 6 7 8 9 10 Pooled SEM

Lipid variable Tristearin Oleate (1%) Oleate (207%) Linoleate (1%) Linoleate (2%) Linolenate (1%) Linolenate (2%7o) Linoleate (1%) + linolenate (1%) (n-3) HUFA 5 (1%) Menhaden oil

Average initial weight (g)3

Weight increase (%)

12

1) ,

Feed efficiency4

Survival (0)

20 18 19 17 19 20 17

3 84 d

383d 334d 524c 587c 630c 626c

0.33e 0.37e 0.37e 0. 56 cd 0.57cd 0.54d 0.53d

36 .7f 5 1.7 ef 56.7de 85.0abc 88.3ab 66.7cde 78.3abc

18 19 17 1

631 c 986 b 1257a 47

0.61c 0.71b 0.80a 0.02

95.0 a 7 3 . 3bcd 90.0ab 6.3

i Values represent means of three replicate groups; 2 means with the same superscript letters are not significantly different (p > 0.05) as determined by Duncan's multiple range test; 3 total weight of 20 fish per group; 4 g gain/g dry feed; 5 highly unsaturated fatty acids of the (n-3) series.

weight gain significantly relative to the tristearin and oleate diets. There was no statistical difference between 1 and 2% levels of a given fatty acid ethyl ester in terms of its effect on weight gain, and the use of both linoleate and linolenate in a single diet did not improve weight gain relative to the use of either one alone. Of the diets containing fatty acid ethyl esters, the one with (n-3) HUFA promoted the greatest weight increase; however, the menhaden oil (control) diet provided a still greater degree of improvement in weight gain. Mortality became evident in fish fed EFAdeficient diets (1-3) from about the fourth week of the experiment and resulted in reduced survival in all three groups (Table 3). As survival decreased in these groups other EFA-deficiency signs began to appear such as erosion of the fins and gill opercles, occasional hemorrhaging at the fin bases, and a ''shock syndrome" manifested as erratic swimming behavior followed by temporary loss of consciousness, which appeared when fish were weighed. The latter sign was observed only in fish fed the diet containing 7% tristearin. Survival ranged from 36.7-95% and did not follow any particular pattern in fish fed diets 4-10. Feed efficiency ranged from 0.33-0.80 (Table 3) and showed the same pattern as weight increase, with the lowest values associated with fish fed tristearin and oleate, inter-

mediate values for fish fed linoleate and linolenate, and highest values for fish fed 1% (n-3) HUFA or menhaden oil. Fatty acid composition Fatty acid composition of whole-body lipids showed similar trends to that of liver and muscle lipids; therefore, only fatty acid composition of whole-body lipids were included in this manuscript. The percentage of total saturates in whole-body polar lipid (Table 4) had a much narrower range than the total dietary saturates, indicating that the tissue saturated fatty acid levels were not strictly a function of diet. Stearic acid (18:0) was not deposited in large amounts in fish fed the 7% tristearin diet and appeared to be largely desaturated to oleic acid (18:1), since the latter fatty acid increased in tissue lipids of fish fed this diet. Whole-body polar monoenes were higher than dietary monoenes for all treatments except the 1 and 2% oleate diets. For these treatments (diets 2 and 3), elevated tissue levels of 18:1 reflected the higher levels of 18:1 in the diets. Levels of (n-6) and (n-3) fatty acids in muscle polar lipid, as well as (n-3) HUFA reflected dietary patterns in general. However, the diet containing (n-3) HUFA had higher levels of eicosapentaenoic acid [20:5(n-3)] than docosahexaenoic acid [22:6 (n-3)], whereas the tissue lipids of fish fed this diet

226 Table 4. Fatty acid composition (% of total fatty acids) of whole-body polar lipid in experiment

11,2,3

Diet number Fatty acid 14:0

16:0

2

3

4

1.6ab

0. 9 bc

0.9bc

20.8ab

8.8d

14.5bcd

1' 0 .6

bc

1 5.4bcd

0 .7 bc

15.1bcd

16:1

8.4

7.4

3.4

7.0

3.9

18:0

8.0b

9.5ab

5.0b

8.3ab

6

18:1

20.5abc

15.0abc

14.labc

15.9abc

10.0b

12.3a

25.2a

.4b

7

8

1 .3bc

1.lbc

0.4c

0.3

22.6a

C

12.1 d

13.2cd

17.3abc

4.8

4.8

4.9

7.2b

8 .3 ab

14.0abc

13.9abc

3. 4 cd

10.3ab

1.Ob

0.7b

11.2a

13.6a

6

.1b

12.9bc 5.0

c

5.4 6.4b

21.5ab

9.0c

2 .8cd

4.3c

1.0b

0.3b

1.0

1.8dc

-4

18:3(n-6)

0.2b

0.lb

2.9a

1.7ab

1.5ab

18:3(n-3)

0.2b

0.3b

0.5b

0. 1 b

2.3b

18:4(n-3)

-

0.1O

4.1a

0.9bc

0.1

20:2(n-6)

0.3

tr5

0.7

0.9

0.3

20:3(n-6)

0 .7 ab

tr

1 .4 ab

0

20:4(n-6)

0.4bc

0.2bc

1.lab

0.3bc

0.1

20:3(n-3)

0.6bcd

0.2cd

3.8a

0.8bcd

2

20:4(n-3)

-

-

0.1

tr

1.Oa

1.4

20:5(n-3)

1.lb

0. 6 b

1.5b

0. 6 b

1.Ob

1.2

22:1

0.6

1.0

0.2

1.0

0.8

0.4

0.7

22:5(n-3)

1.6bc

1.Ocd

-

1 .2 bcd

1.2bcd

0 .8 d

1.Ocd

2.6a

22:6(n-3)

4 .1bc

4 .7bc

1.5c

3.5C

2.8c

2.8c

3.6c

4 .8bc

24.2 23.6 16.0 8.3 4.9 27.9

21.2 20.4 8.2 20.0 4.8 30.2

Totals Saturates Monoenoics (n-6) (n-3) 6 (n-3) HUFA Other 7

26.5 30.2 4.5 7.6 6.8 31.2

33.3 36.5 2.2 6.9 6.3 21.1

c

20.9 25.4 6.0 11.3 2.9 36.4

23.9 23.8 13.6 7.1 5.3 31.6

1.8

a

c

.5ab

2.4a

15.8bC

8.8

2 .9cd

.8ab

ce

12.7a

18:2(n-6)

c

10

9

6

5'*

b

4.0b

0.3b

2. 6 b

l.Obc

2.0abc

0.6bc

1.Obc

2.6ab

0.6

0.1

0.9

0.1

0.9

1 .Oab

0 .6 ab

0 .6 ab

0 .2 ab

1 .4 ab

0.7bc

0. 6 bc

0.2bc

0. 6 bc

1.6a

1.7bcd

2.lbc

1I.bcd

0.2cd

-

0 .5 b

0.2bc

0.4bc

1.4a

l.lb

0.6b

5.3

a b

22.6 20.9 5.5 23.8 5.7 27.2

a

26.3 19.8 13.0 13.9 8.0 27.0

a

1.1

0.1 1.8 b 10.6

4.2

a

36.5 31.0 4.1 19.4 17.7 9.0

1 .5bcd 8 .lab

29.3 19.6 9.1 20.3 13.7 21.7

I Means of three composite samples except where indicated: (*) = means of two composite samples; () = one sample only. Pooled SEM for each fatty acid was: 14:0, 0.29; 16:0, 1.83; 16:1, 1.56; 18:0, 1.24; 18:1, 3.08; 18:2(n-6), 0.63; 18:3(n-6), 0.45; 18:3(n-3), 1.31; 18:4(n-3), 0.63; 20:2(n-6), 0.34; 20:3(n-6), 0.45; 20:4(n-6), 0.25; 20:3(n-3), 0.51; 20:4(n-3), 0.12; 20:5(n-3), 0.47; 22:1, 0.47; 22:5(n-3), 0.21; 22:6(n-3), 1.15; 2 means with the same letters are not significantly different (p>0.05) as determined by Duncan's multiple range test; 3 lipid variables of the diets were as follows (diet no., lipid variable): 1, tristearin; 2, oleate(l%); 3, oleate(2%); 4, linoleate(l%); 5, linoleate(2%); 6, linolenate(l °); 7, linolenate(2%); 8, linoleate(1%)+ linolenate(l%); 9, (n-3) HUFA(1%7); 10, menhaden fish oil; 4 - = not detected; 5 tr = trace (

Essential fatty acid requirement of juvenile red drum (Sciaenops ocellatus).

Feeding experiments and laboratory analyses were conducted to establish the essential fatty acid (EFA) requirement of red drum (Sciaenops ocellatus). ...
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